Corrigendum: Phytohormone and Putative Defense Gene Expression Differentiates the Response of ‘Hayward’ Kiwifruit to Psa and Pfm Infections

Wurms, Kirstin; Hardaker, Allan J.; Chee, A. Ah; Bowen, Judith L.; Phipps, Janet; Taylor, J.; Jensen, Dwayne J.; Cooney, Janine M.; Wohlers, Mark; Regliński, T.

doi:10.3389/fpls.2017.02012

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“…This low efficiency of targeted integration through Agrobacterium -mediated transformation was previously reported [24,27,41] and could be related to the low number of T-DNA copies delivered into the plant cells. Strategies based on particle bombardment or DNA virus-based replicons were reported to be valuable for HDR-mediated gene replacement in many plant species such as rice, maize, soybean, wheat, cotton and tomato, for reviews see [28,52]. Nevertheless, those direct DNA transfer techniques or genetically modified viruses are not necessarily accessible in all laboratories and delivery of DNA into the cell via Agrobacterium mediated transformation is still the method of choice for a great number of plants, including tomato.…”

Section: Discussionmentioning

confidence: 99%

The DFR locus: A smart landing pad for targeted transgene insertion in tomato

et al. 2018

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Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.

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